(1) Field of the Invention
This invention concerns a method for preparing a highly purified glycan extract, as well as the purified glycan product.
(2) Description of the Art
Bacillus Calmette Guerine (BCG) vaccine is an attenuated form of Mycobacterium bovis that has been used as tuberculosis vaccine for more than 70 years. BCG has proved to be a general immunostimulant and intravesicular BCG immunotherapy is currently an approved and effective clinical intervention against superficial bladder cancer, providing long-term protection from tumour recurrence, progression and mortality. BCG has also been demonstrated to be effective against other tumours such as melanoma, sarcoma, lung cancer and leukemia. Success in the treatment of these tumours with BCG has proved to be variable since direct contact between BCG and the tumour cells is apparently required.
The mode of action of BCG against bladder carcinoma is not well understood, but activity has been attributed to a nonspecific stimulation of the lymphoreticuloendothelial system. BCG vaccine consists of a mixture of dead and living whole organisms with associated cell debris and approximately 5% of cancer patients treated with BCG experience adverse reactions to the therapy associated with the use of viable bacteria. These reactions include mild fever, urethritis and, in extreme situations, disseminated mycobacterioses. Thus, it is preferable to identify and isolate non-viable bacterial components of BCG with anticancer activity which could provide material with potential clinical application.
Hardham & James (1980) reported the presence of a covering material on the surface of the Glaxo substrain of BCG vaccine. This observation was shown to be more general for other BCG substrains and accounted for the lack of effect of wetting agents on the state of dispersion. The composition of the interfacial integument appears to be substantially polysaccharidic although cell-surface proteins account for the major part of cellular surface charges. Rastogi et al., (1986) found polysaccharide-rich outer layers in 18 mycobacterial species but Kristensen et al. (1992) showed that the cationic charge of BCG could be abolished with proteolytic enzymes, suggesting that it was essentially due to surface protein. The anionic charge, on the other hand, was provided by carboxylic acids, phosphoesters and strong acidic groups such as sulfate. Accordingly, it was of interest to note that the cellular integument could be removed with bacterial pronase, apparently without killing the cells. Electron microscopy and light microscopy demonstrated that aggregates of BCG cells were often covered by variable amounts of the integument. A more detailed study suggested that the integument separated by pronase was substantially carbohydrate in composition. The integument was patchy in appearance and of variable thickness, leading to the suggestion that growth of integument may be provoked by environmental stresses such as oxygen tension. Klegerman et al. (1996) recently reported that the electron transparent zone associated with the integument varied in thickness from 1-250 nm and suggested that it may be the source of complex glycans extracted from BCG from boiling water. These glycans, accounting for as much as 12% of the dry cell weight of the Tice.RTM. BCG substrain, were shown to possess considerable antineoplastic activity. Of the mixed glycans separated by boiling water extraction from the Tice.RTM. substrain of BCG vaccine, one glucan, PS1 A1, was shown to have the highest specific activity against a murine sarcoma model in vivo but no in vitro activity was detected against a wide variety of tumour models, indicating an immunostimulant mechanism (Wang et al., 1995).
Various methods have been developed for obtaining extracts of glycans and other popolysaccharides from various sources such as bacteria, yeast and plants. Furthermore, it has previously been demonstrated that complex high molecular weight antineoplastic glycans can be extracted by boiling intact cells of Mycobacteria, especially M. bovis (BCG vaccine), M. vaccae and M. phlei. Despite these advances in processes for obtaining therapeutic glycans from various natural sources, there remains a need for processes capable of producing glycan extracts in high purities and yields.